Tumor Promoter 12-O-tetradecanoylphorbol-13-acetate Research Articles

Abstract 2480: Role of Stat1 in epithelial carcinogenesis

Abstract Aberrant expression and/ or activity of STATs 1, 3, and 5 have been reported in multiple human cancers as well as in murine models of tumorigenesis. Previous work performed in our lab demonstrated a critical role for Stat3 in mediating the initiation and promotion stages of epithelial multi-stage carcinogenesis in mouse skin. In light of these findings, we have begun investigating the role of other STATs, particularly Stat1, in multistage skin carcinogenesis using both wild-type and Stat1 knockout (KO) mice. Western blot analyses revealed that topical application of the skin tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and chrysarobin induced phosphorylation of Stat1 in mouse epidermis. In addition, following treatment with chrysarobin, the level of total Stat1 protein was also dramatically elevated. To further investigate the role of Stat1 during epithelial carcinogenesis, Stat1 KO mice and wild-type littermates were initiated with 25 nmol DMBA and promoted with either 6.8 nmol TPA or 220 nmol chrysarobin. No significant differences were observed in either tumor incidence or multiplicity when TPA was used as the promoting agent, suggesting that Stat1 does not play a significant role in TPA-mediated skin tumor promotion. In contrast, Stat1 KO mice exhibited a dramatic reduction in both tumor multiplicity and incidence as compared to wild-type littermates when chrysarobin was used as the promoting agent. Additionally, following topical treatment with chrysarobin, Stat1 KO mice had a reduced epidermal proliferative response. To further investigate the impact of Stat1 deletion on chrysarobin-induced tumor promotion, Stat1 KO mice and wild-type controls were treated topically with chrysarobin and the inflammatory response was assessed. Stat1 KO mice displayed a significant reduction in Cox-2 induction compared to wild-type controls. Preliminary findings also showed that Stat1 KO mice have reduced immune cell influx following chrysarobin treatment. To investigate the potential role of reactive oxygen species (ROS) in the effects of chrysarobin on Stat1 activation, the dorsal skin of FVB mice were pre-treated topically with the anti-oxidants ascorbyl palmitate and α-tocopherol acetate. Topical application of α-tocopherol acetate significantly attenuated the epidermal proliferative response and Stat1 activation suggesting that chrysarobin-mediated generation of ROS may play a role in its effects on Stat1 during tumor promotion. Additional studies are underway to further determine the mechanistic basis for the role of Stat1 in chrysarobin-mediated tumor promotion. Supported by NIH grant CA 76520. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2480.

Abstract 2483: Transgenic mice to evaluate the role of S-adenosylmethionine decarboxylase in skin carcinogenesis

Abstract To evaluate the role of S-adenosylmethionine decarboxylase (AdoMetDC) and altered polyamine levels in mouse skin carcinogenesis, we utilized the tetracycline-regulated system to produce conditional expression of AdoMetDC in basal keratinocytes. AdoMetDC is a key enzyme in polyamine biosynthesis and it catalyzes the decarboxylation reaction converting AdoMet to dcAdoMet. dcAdoMet provides the aminopropyl groups for the synthesis of the higher polyamines spermidine and spermine from the precursor putrescine. Therefore, elevated AdoMetDC activity may promote the synthesis of higher polyamines at the expense of putrescine, and increased putrescine levels are tightly linked to tumor promotion in mouse skin. AdoMetDC activity is also known to be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). To make the DNA construct for microinjection, human AdoMetDC cDNA was inserted into the TetO vector followed by an internal ribosome entry site and luciferase coding sequence. Potential founder mice were crossed to mice with skin-specific expression of tetracycline-regulated transcriptional activator (tTA) directed by bovine keratin 5 (K5) promoter elements to generate K5-tTA/TetO-AdoMetDC bitransgenic mice. Transgene expression has been characterized by in vivo bioluminescent imaging of luciferase activity, in vitro luciferase activity as well as AdoMetDC activity assay and Western blotting in 7 week old mice. In bitransgenic mice, AdoMetDC is constitutively expressed in the skin and expression can be repressed by exposure to the tetracycline analogue doxycycline (Dox). In untreated bitransgenic mice generated from two founder lines, we saw a massive increase in luciferase activity and these mice have a thin fur phenotype. Minimal transgene expression is detected in either single transgenic TetO-AdoMetDC or Dox-treated bitransgenic mice. Untreated bitransgenic mice from one founder line exhibit a 7.2- fold increase in AdoMetDC activity in epidermis, a 7.8-fold increase in dermis, and dcAdoMet levels are also elevated by 8.0-fold in dermis. Effects of AdoMetDC overexpression on TPA induced polyamine biosynthesis and susceptibility to 7,12-dimethylbenz(a)anthracene/TPA carcinogenesis are now being investigated. These mice with regulated AdoMetDC expression in the skin provide a valuable model system to evaluate the role of AdoMetDC and polyamines in skin carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2483.

Abstract 1583: Plumbagin, a medicinal plant-derived naphthoquinone with both chemopreventive and therapeutic properties, selectively inhibits expression of Protein Kinase Cε, a predictive biomarker of prostate cancer progression

Abstract PKC is a major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). PKC represents a large family of phosphatidylserine-dependent serine/threonine kinases. Protein kinase Cε (PKCε) is a member of the novel PKC sub-family. PKCε is a transforming oncogene and a predictive biomarker of various human cancers, including prostate cancer (PCa). We have shown that PKCε expression level correlates with human PCa aggressiveness. PKCε overexpression is sufficient to transform androgen-dependent LNCaP cells into an androgen-independent variant, which rapidly initiates growth in vivo in both intact and castrated male nude mice. PKCε silencing using specific siRNA in DU145 cells inhibits Stat3-regulated gene expression (COX-2) and cell invasion. Also, PKCε deletion (even one allele) in TRAMP mice prevents development of PCa. Taken together, the evidence indicates an association of PKCε to PCa development and aggressiveness. Thus, PKCε may be a potential molecular target for prevention and treatment of PCa. To screen for PKCε inhibitors, we evaluated by Western Blot analyses 19 plant-derived chemopreventive agents and found plumbagin (PL) to be a selective inhibitor of PKCε expression. In this experiment, human prostate DU145 cells at 70% to 80% confluency were serum starved for 24 hr. Cells were treated with various natural agents: curcurmin (50μM), capsaicin (50μM), silibinin (200μM), epigallocatechin gallate (25μM), resveratrol (50μM), diallyl sulfide (50μM), lycopene (10μM), apigenin (10μM), docosahexacnoic acid (10μM), caffeic acid (25μM), genistein (10μM), tocopherol (10μM), β-carotene (10%), guggul (50μM), PL (50μM), ursodeoxycholic acid (50μM) in DMSO (final concentration, 0.1%) for 6 hr. Whole-cell lysates were prepared and used for Western blot analysis of PKC isoforms (α, βI, βII, δ, ε, γ, η, μ. ζ). Proteins, normalized to β-actin, were quantitated and only PL inhibited PKCε expression. In a more detailed dose-response study (5-20μM) with PL determined 10μM as IC50 for PKCε. Treatment of PCa cells (DU145 and CW22Rv1) with PL inhibits activation of transcription factors (AP-1, Stat3 and NF-kB), cell invasion and PCa cell xenografts in nude mice (J Biol Chem 2006, 281:17023; Cancer Res. 2008, 68:9024). PL (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone, was isolated from the roots of the Plumbago zeylanica L. (also known as Chitrak;). The roots of Plumbago zeylanica have been used in Indian medicine for more than 2,500 years for treatments of various ailments. PL is also present in black walnut and other various medicinal plants. We conclude that PL may be explored as a naturally occurring agent in the prevention and treatment of prostate cancer (Support: NIH grant CA35368). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1583.

Transforming growth factor β1 enhances tumor promotion in mouse skin carcinogenesis

Transforming growth factor beta1 (TGFbeta1) expression is elevated by tumor promoters in the mouse skin, but its role in tumor promotion has not been well defined. To investigate this, we have compared TGFbeta1+/+ and +/- mice in a two-stage skin chemical carcinogenesis protocol. Surprisingly, TGFbeta1+/- mice had fewer number and incidence of benign papillomas, reduced epidermal and tumor cell proliferation and reduced epidermal TGFbeta1 and nuclear p-Smad2 localization in response to the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) compared with TGFbeta1+/+ mice. Maximal TPA activation of protein kinase C (PKCalpha) as measured by activity assays and activation of target genes and induction of cornified envelopes correlated with TGFbeta1 gene dosage in keratinocytes and addition of exogenous TGFbeta1 restored the cornification defect in TGFbeta1+/- keratinocytes. Similarly, inhibition of ALK5-suppressed TPA-mediated PKCalpha activation suggesting that physiological levels of TGFbeta1 are required for maximal activation of PKC-dependent mitogenic responses. Paradoxically, the TPA-induced inflammatory response was greater in TGFbeta1+/- skin, but TGFbeta1+/+ papillomas had more tumor infiltrating myeloperoxidase-positive cells and pro-inflammatory gene expression was elevated in v-ras(Ha)-transduced TGFbeta1+/+ but not TGFbeta1+/- keratinocytes. Thus, ras activation switches TGFbeta1 to a pro-inflammatory cytokine. Despite this differential proliferative and inflammatory response to TPA and enhanced papilloma formation in the TGFbeta1+/+ mice, the frequency of malignant conversion was reduced compared with TGFbeta1+/- mice. Therefore, TGFbeta1 promotes benign tumors by modifying tumor promoter-induced cell proliferation and inflammation but retains a suppressive function for malignant conversion.

In vivo measurement of epidermal thickness changes associated with tumor promotion in murine models

The characterization of tissue morphology in murine models of pathogenesis has traditionally been carried out by excision of affected tissues with subsequent immunohistological examination. Excision-based histology provides a limited two-dimensional presentation of tissue morphology at the cost of halting disease progression at a single time point and sacrifice of the animal. We investigate the use of noninvasive reflectance mode confocal scanning laser microscopy (rCSLM) as an alternative tool to biopsy in documenting epidermal hyperplasia in murine models exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). An automated technique utilizing average axial rCSLM reflectance profiles is used to extract epidermal thickness values from rCSLM data cubes. In comparisons to epidermal thicknesses determined from hematoxylin and eosin (H&E) stained tissue sections, we find no significant correlation to rCSLM-derived thickness values. This results from method-specific artifacts: physical alterations of tissue during H&E preparation in standard histology and specimen-induced abberations in rCSLM imaging. Despite their disagreement, both histology and rCSLM methods reliably measure statistically significant thickness changes in response to TPA exposure. Our results demonstrate that in vivo rCSLM imaging provides epithelial biologists an accurate noninvasive means to monitor cutaneous pathogenesis.

A Genetic Variant of Aurora Kinase A Promotes Genomic Instability Leading to Highly Malignant Skin Tumors

Aurora kinase A (Aurora-A) belongs to a highly conserved family of mitotis-regulating serine/threonine kinases implicated in epithelial cancers. Initially we examined Aurora-A expression levels at different stages of human skin cancer. Nuclear Aurora-A was detected in benign lesions and became more diffused but broadly expressed in well and poorly differentiated squamous cell carcinomas (SCC), indicating that Aurora-A deregulation may contribute to SCC development. To mimic the overexpression of Aurora-A observed in human skin cancers, we established a gene-switch mouse model in which the human variant of Aurora-A (Phe31Ile) was expressed in the epidermis upon topical application of the inducer RU486 (Aurora-AGS). Overexpression of Aurora-A alone or in combination with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), did not result in SCC formation in Aurora-AGS mice. Moreover, Aurora-A overexpression in naive keratinocytes resulted in spindle defects in vitro and marked cell death in vivo, suggesting that the failure of Aurora-A to initiate tumorigenesis was due to induction of catastrophic cell death. However, Aurora-A overexpression combined with exposure to TPA and the mutagen 7,12-dimethylbenz(a)anthracene accelerated SCC development with greater metastatic activity than control mice, indicating that Aurora-A cannot initiate skin carcinogenesis but rather promotes the malignant conversion of skin papillomas. Further characterization of SCCs revealed centrosome amplification and genomic alterations by array CGH analysis, indicating that Aurora-A overexpression induces a high level of genomic instability that favors the development of aggressive and metastatic tumors. Our findings strongly implicate Aurora-A overexpression in the malignant progression of skin tumors and suggest that Aurora-A may be an important therapeutic target.

2- O-β- d-Glucopyranosyl- sn-glycerol based analogues of sulfoquinovosyldiacylglycerols (SQDG) and their role in inhibiting Epstein-Barr virus early antigen activation

New sulfoquinovosyldiacylglycerols derived from 2- O-β- d-glucopyranosyl- sn-glycerol, carrying acyl chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure in which a sulfonate is introduced at the C-6 position of glucose by oxidation of a thioacetate in the presence of the unprotected secondary hydroxyl groups, and tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. Our study has allowed to ascertain the role of the 6′-sulfonate group and the need of a free hydroxyl group on the glycerol moiety in inhibiting the EBV activation promoted by the tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA).

A Simple Analogue of Tumor-Promoting Aplysiatoxin Is an Antineoplastic Agent Rather Than a Tumor Promoter: Development of a Synthetically Accessible Protein Kinase C Activator with Bryostatin-like Activity

Protein kinase C (PKC) is widely recognized as a therapeutic target in intractable diseases such as cancer, Alzheimer's disease (AD), and acquired immune deficiency syndrome (AIDS). While inhibition of PKC is a general therapeutic strategy for the treatment of cancer, PKC activators are potential therapeutic agents for AD and AIDS. However, concerns have been raised about their therapeutic use since PKC activators such as phorbol esters exhibit potent tumor-promoting activities. Naturally occurring bryostatin 1 (bryo-1), prostratin, and 12-deoxyphorbol 13-phenylacetate (DPP) are fascinating PKC activators without tumor-promoting activities. Bryo-1 is currently in clinical trials for the treatment of cancer and is also effective against AD. Prostratin and DPP are attractive candidates for the adjunctive treatment of human immunodeficiency virus (HIV) infection. However, their limited availability from natural sources and synthetic complexity have hampered further development as therapeutic agents. We report here easy access (22 steps) to a simple analogue (1) of the tumor-promoting aplysiatoxin (ATX) as a novel PKC activator with anticancer and anti-tumor-promoting activities. Anticancer activities of 1 against several human cancer cell lines were comparable to those of bryo-1. Moreover, 1 as well as bryo-1 significantly inhibited the Epstein-Barr virus early antigen (EBV-EA) induction by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), whereas ATX strongly induced EBV-EA. This inhibitory effect is characteristic of antitumor promoters. Compound 1 as well as bryo-1 displayed significant binding and activation of PKCdelta and induced its translocation to the nuclear membrane in CHO-K1 cells. This study provides a synthetically accessible PKC activator with bryo-1-like activities, which could be another therapeutic lead for cancer, AD, and AIDS.

Inhibition of NF-κB signaling interferes with phorbol ester-induced growth arrest of keratinocytes in a TNFR1-independent manner

A skin-specific block in NF-κB signaling leads to hyperproliferation of the keratinocytes, inflammation, and spontaneous development of squamous cell carcinoma (SCC). Here we show that an inhibition of NF-κB signaling in keratinocytes, via the expression of the super-repressor/ degradation-resistant form of the IκBα protein (IκBαDN), interferes with the growth arrest induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). The IκBαDN cells are able to overcome the TPA-induced cell cycle block. Although SCC development as well as hyperproliferation due to IκBαDN expression in keratinocytes is known to require TNFR1 signaling, the effect of IκBαDN on phorbol ester signaling is downstream/independent of TNFR1. These data thus identify an interaction between IκBαDN and the tumor promoter TPA in the growth regulation of keratinocytes. The proposed mechanism is also likely to be significant in the process of cancer development due to NF-κB inhibition.

A novel Src kinase inhibitor reduces tumour formation in a skin carcinogenesis model

The Src family tyrosine kinases are key modulators of cancer cell invasion and metastasis and a number of Src kinase inhibitors are currently in clinical development for the treatment of solid tumours. However, there is growing evidence that Src is also upregulated at very early stages of epithelial cancer development. We have investigated the role of Src in mouse skin, which is one of the most tractable models of epithelial homoeostasis and tumorigenesis. We found that Src protein expression and activity was regulated during the normal hair cycle and was increased specifically during the proliferative anagen phase and also in response to the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). AZD0530, a selective Src inhibitor, prevented the TPA-induced proliferation of basal keratinocytes both in vivo and in vitro. Moreover, treatment with AZD0530 reduced papilloma formation following the well-established 7,12-dimethylbenz(a)anthracene/TPA skin carcinogenesis protocol but did not inhibit the subsequent proliferation of the papillomas. Furthermore, AZD0530 did not alter the malignant conversion of papillomas to squamous cell carcinoma suggesting a role for Src in early tumour development in the skin carcinogenesis model, rather than at later stages of tumour progression. Src expression and activity were also seen in human actinic keratoses that are hyperproliferative pre-malignant skin lesions, indicating that Src may also play a role in the early stages of human skin tumour development. Thus, Src inhibitors such as AZD0530 may therefore have chemopreventative properties in patients with hyperproliferative epidermal disorders.

Abstract B72: Cancer chemopreventive potential of henna

Abstract B72 “Henna” is a powder of the leaves of Lawsonia inermis L. This powder is used mainly as a pigment for coloring skin, hair, fingernails, leather, silk and wool. The anticancer potential of henna powder was evaluated using in vitro and in vivo assays. Henna inhibited tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) - induced activation of Epstein-Barr virus early antigen in lymphoblastoid Raji cells. This indicated the anticancer potential of henna powder. The activity of henna powder was also studied in the ultraviolet B light (280-320 nm wavelength, 34.3 kJ/m2) - initiated and TPA- promoted two stage mouse skin carcinogenesis model. Topical application of a suspension of henna (0.5 mg/ml acetone) decreased tumor incidence in this model system by 70% and tumor multiplicity by 40% at ten weeks of treatment. Lawsone and its precursors 1, 2, 4-trihydroxynaphthalene-1,4-di-beta-glucopyranoside and 1, 2-dihydroxy-4-O-glucosyloxynaphthalene may be the pharmacologically active compounds in henna. In the present study lawsone inhibited TPA-induced activation of Epstein-Barr virus early antigen by 94%. The cancer inhibitory effect of topically applied henna powder was further demonstrated in the peroxynitrite (PN) -initiated and TPA - promoted two stage mouse skin carcinogenesis model system. In this model system, henna decreased tumor incidence by 55% and tumor multiplicity by 57% at 11 weeks of treatment. These data suggest that henna powder as well as its major coloring pigment, lawsone and/or its precursors may be useful as cancer preventive agents. The ability of lawsone and its precursors to perturb electron transfer reactions and free radical processes may partially account for their cancer preventive effects. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B72.

Inhibitory effects of oligonol on phorbol ester-induced tumor promotion and COX-2 expression in mouse skin: NF-κB and C/EBP as potential targets

Plant polyphenols possess anti-oxidant and anti-inflammatory activities and are hence potential candidates for preventing cancer. The present study was aimed at evaluating the anti-inflammatory and anti-tumor promoting activity of oligonol, a formulation of catechin-type oligomers, in mouse skin stimulated with a proto-type tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment of mouse skin with oligonol significantly inhibited TPA-induced expression of cyclooxygenase-2 (COX-2). Oligonol diminished nuclear translocation and DNA binding of nuclear factor-kappaB (NF-κB) via blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Moreover, oligonol suppressed TPA-induced DNA binding of CCAAT/enhancer-binding protein (C/EBP) in mouse skin. Oligonol pretreatment also attenuated the phosphorylation and/or catalytic activities of extracellular signal-regulated protein kinase-1/2 (ERK1/2) and p38 mitogen-activated protein (MAP) kinase. Moreover, p38 MAP kinase inhibitor SB203580, but not the MEK inhibitor U0126, negated TPA-induced DNA binding of C/EBP. In addition, oligonol reduced the incidence and the multiplicity of papillomas and squamous cell carcinomas in 7,12-dimethylbenz[ a]anthracene (DMBA)-initiated and TPA-promoted mouse skin, and prolonged the survival of tumor-bearing mice. Pretreatment with oligonol diminished the levels of proliferating cell nuclear antigen and expression of COX-2 in papillomas and carcinomas, respectively, as compared to DMBA plus TPA treatment alone. Taken together, the above findings suggest that oligonol inhibits TPA-induced COX-2 expression by blocking the activation of NF-κB and C/EBP via modulation of MAP kinases and suppresses chemically induced mouse skin tumorigenesis.

Podoplanin Is a Novel Fos Target Gene in Skin Carcinogenesis

Expression and function of the oncogenic transcription factor activator protein (AP-1; mainly composed of Jun and Fos proteins) is required for neoplastic transformation of keratinocytes in vitro and tumor promotion as well as malignant progression in vivo. Here, we describe the identification of 372 differentially expressed genes comparing skin tumor samples of K5-SOS-F transgenic mice (Fos(f/f) SOS(+)) with samples derived from animals with a specific deletion of c-Fos in keratinocytes (Fos(Deltaep) SOS(+)). Fos-dependent transcription of selected genes was confirmed by quantitative real-time PCR analysis using tumor samples and mouse back skin treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). One of the most differentially expressed genes encodes the small mucin-like glycoprotein Podoplanin (Pdpn), whose expression correlates with malignant progression in mouse tumor model systems and human cancer. We found Pdpn and Fos expression in chemically induced mouse skin tumors, and detailed analysis of the Pdpn gene promoter revealed impaired activity in Fos-deficient mouse embryonic fibroblasts, which could be restored by ectopic Fos expression. Direct Fos protein binding to the Pdpn promoter was shown by chromatin immunoprecipitation and a TPA-induced complex at a TPA-responsive element-like motif in the proximal promoter was identified by electrophoretic mobility shift assays. In summary, we could define a Fos-dependent genetic program in a well-established model of skin tumors. Systematic analysis of these novel target genes will guide us in elucidating the molecular mechanisms of AP-1-regulated pathways that are critically implicated in neoplastic transformation and/or malignant progression.

Modulation of PKC delta and epsilon distribution by plant phenols in mouse epidermis

Polymeric black tea polyphenols (PBPs) have been shown to possess anti-tumor-promoting effects in two-stage skin carcinogenesis. However, their mechanisms of action are not fully elucidated. In this study, mechanisms of PBP-mediated antipromoting effects were investigated in a mouse model employing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Compared to controls, a single topical application of TPA to mouse skin increased the translocation of protein kinase C (PKC) from cytosol to membrane. Pretreatment with PBPs 1–3 decreased TPA-induced translocation of PKC isozymes (α, β, η, γ, ε) from cytosol to membrane, whereas PBPs 4 and 5 were less effective. The levels of PKCs δ and ζ in cytosol/membrane were similar in all the treatment groups. Complementary confocal microscopic evaluation showed a decrease in TPA-induced PKCα fluorescence in PBP-3-pretreated membranes, whereas pretreatment with PBP-5 did not show a similar decrease. Based on the experiments with specific enzyme inhibitors and phosphospecific antibodies, both PBP-3 and PBP-5 were observed to decrease TPA-induced level and/or activity of phosphatidylinositol 3-kinase (PI3K) and AKT1 (pS473). An additional ability of PBP-3 to inhibit site-specific phosphorylation of PKCα at all three positions responsible for its activation [PKCα (pT497), PKC PAN (βII pS660), PKCα/βII (pT638/641)] and AKT1 at the Thr308 position, along with a decrease in TPA-induced PDK1 protein level, correlated with the inhibition of translocation of PKC, which may impart relatively stronger chemoprotective activity to PBP-3 than to PBP-5. Altogether, PBP-mediated decrease in TPA-induced PKC phosphorylation correlated well with decreased TPA-induced NF-κB phosphorylation and downstream target proteins associated with proliferation, apoptosis, and inflammation in mouse skin. Results suggest that the antipromoting effects of PBPs are due to modulation of TPA-induced PI3K-mediated signal transduction.

Anti-inflammatory effects of Asparagus cochinchinensis extract in acute and chronic cutaneous inflammation

Asparagus cochinchinensis Merrill (Liliaceae) is a traditional herbal medicine used for treatment of various diseases [1, 2]. In the present study, we determine the ability of Asparagus cochinchinensis extract (ACE) to inhibit skin inflammation following exposure to the well-characterized protein kinase C activator and tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) [3]. ACE ameliorate the inflammatory phenotype, leading to substantial reductions in skin thickness (54.3% inhibition) and tissue weight (32.5% inhibition), inflammatory cytokine production, neutrophil-mediated myeloperoxidase activity, and various histopathological indicators (p<0.01). ACE also significantly inhibits phorbol ester–induced irritant contact dermatitis and increases acetic acid–induced vascular permeability (p<0.05). ACE is also effective at reducing inflammatory damage induced by chronic TPA exposure (p<0.01). These results demonstrate that ACE is an effective anti-inflammatory agent in murine phorbol ester–induced dermatitis, and suggest that the compound may have therapeutic potential in a variety of immune-related cutaneous diseases.

Pterostilbene is equally potent as resveratrol in inhibiting 12-O-tetradecanoylphorbol-13-acetate activated NFκB, AP-1, COX-2, and iNOS in mouse epidermis

Resveratrol, a phytoalexin present in grapes, has been reported to inhibit multistage mouse skin carcinogenesis. Recent studies showed that topically applied resveratrol significantly inhibited cyclooxygenase-2 (COX-2) expression and activation of nuclear factor-kappaB (NF-kappaB) induced by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis. The aim of the present study was to further explore the effect of resveratrol on TPA-induced signaling pathways in mouse epidermis and to compare with its dimethylether, pterostilbene. Resveratrol and pterostilbene significantly reduced activator protein 1 (AP-1) and NF-kappaB activation. In the case of AP-1, the binding of c-Jun subunit was particularly affected, while only slight effect on c-Fos binding to TPA-responsive element (AP-1 binding consensus sequence) (TRE) site was observed. Both stilbenes inhibited the activation of NF-kappaB by blocking the translocation of p65 to the nucleus and increasing the retention of IkappaBa in the cytosol. The latter might be related to decreased activity of IkappaB kinase and/or proteasome 20S. Reduced activation of transcription factors decreased the expression and activity of COX-2 and inducible nitric oxide synthase (iNOS). In most assays, pterostilbene was either equally or significantly more potent than resveratrol. Pterostilbene might show higher biological activity due to its possible better bioavailability, since substitution of hydroxy with methoxy group increases lipophilicity.

Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage.

Chronic exposure to 12-O-tetradecanoylphorbol-13-acetate represses sod2 induction in vivo: the negative role of p50

It is well documented that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) can activate manganese superoxide dismutase (MnSOD) expression. However, it is unclear how repeated exposure to TPA following a single application of tumor initiator 7,12-dimethylbenz-(a)-anthracene causes tumor development. We generated transgenic mice expressing human MnSOD promoter- and enhancer-driven luciferase reporter gene and used a non-invasive imaging system to investigate the effects of TPA on MnSOD expression in vivo. Our data indicate that TPA initially activates MnSOD expression, but this positive effect declines after repeated applications. Changes in MnSOD expression in vivo were verified by measuring MnSOD mRNA and protein levels. Using chromatin immunoprecipitation coupled to Western analysis of the transcription factors known to be essential for the constitutive and TPA-induced transcription of MnSOD, we found that TPA treatment leads to both activation and inactivation of MnSOD gene transcription. During the activation phase, the levels of p50, p65, specificity protein 1 (Sp1) and nucleophosmin (NPM) increase after TPA treatments. Sustained treatments with TPA lead to further increase of p50 but not p65, Sp1 or NPM, suggesting that excess p50 may have inhibitory effects leading to the suppression of MnSOD. Alteration of p50 levels by expressing p50 cDNA or p50 small interfering RNA in mouse epithelial (JB6) cells confirms that p50 is inhibitory to MnSOD transcription. These findings identify p50 as having a negative effect on MnSOD induction upon repeated applications of TPA and provide an insight into a cause for the reduction of MnSOD expression during early stages of skin carcinogenesis.

Protein kinase C epsilon signals ultraviolet light-induced cutaneous damage and development of squamous cell carcinoma possibly through Induction of specific cytokines in a paracrine mechanism.

Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases, is not only the major intracellular receptor for the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) but also is activated by a variety of stress factors including ultraviolet radiation (UVR). PKCepsilon is among six isoforms (alpha, delta, epsilon, eta, mu and zeta) expressed in the mouse skin. To determine the in vivo functional specificity of PKCepsilon in mouse skin carcinogenesis, we generated PKCepsilon transgenic mouse (FVB/N) lines 224 and 215 that overexpress PKCepsilon protein approximately 8- and 18-fold, respectively, over endogenous levels in the basal epidermal cells and cells of the hair follicle. PKCepsilon transgenic mice were observed to be highly sensitive to the development of papilloma-independent metastatic squamous cell carcinoma (mSCC) elicited either by repeated exposure to UVR or by the 7,12-Dimethylbenzanthracene-TPA tumor promotion protocol. The development of squamous cell carcinoma (SCC) appears to be linked to the PKCepsilon-mediated induction of cytokine tumor necrosis factor-alpha(TNFalpha). Immunohistochemical analysis for the expression of PKCepsilon in the SCC of PKCepsilon transgenic mice revealed that PKCepsilon was not expressed in the tumor itself; however, the uninvolved tissue surrounding the SCC exhibited intense PKCepsilon expression. Also, human SCC, similar to mouse SCC, did not express PKCepsilon in the tumor, whereas the surrounding uninvolved epidermis revealed strong PKCepsilon expression. These findings in both the PKCepsilon mouse model and human SCC indicate that overexpression of PKCepsilon in epidermis may lead to a microenvironment, which is suitable for enhancing the development of mSCC by a paracrine mechanism involving specific cytokines including TNFalpha.

Cancer Chemopreventive Effects of Cycloartane-Type and Related Triterpenoids in in Vitro and in Vivo Models

Forty-eight natural and semisynthetic cycloartane-type and related triterpenoids have been evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells as a primary screening test for anti-tumor promoters. In addition, these triterpenoids have been tested for their inhibitory effects on activation of (+/-)-(E)-methtyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexemide (NOR 1), a nitric oxide (NO) donor, as a primary screening test for anti-tumor initiators. All of the compounds tested exhibited inhibitory effects on both EBV-EA and NOR 1 activation. Six of these compounds having a C-24 hydroxylated side chain, viz., (24R)-cycloart-25-ene-3beta,24-diol (9), (24R)-cycloartane-3beta,24,25-triol (11), (24S)-cycloartane-3beta,24,25-triol (12), (24xi)-24-methylcycloartane-3beta,24,241-triol (14), (24xi)-241-methoxy-24-methylcycloartane-3beta,24-diol (15), and (24xi)-24,25-dihydroxycycloartan-3-one (27), showed higher inhibitory effects than the others tested on both EBV-EA (IC50 values of 6.1-7.4 nM) and NOR 1 activation. Furthermore, compounds 14 and 15 exhibited inhibitory effects on skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.